Regulation of GLUT1, the basal glucose transporter of skeletal muscle and the predominant dehydroascorbic acid (DHA) transporter, has not been fully-elucidated in terms of factors that influence GLUT1 cell surface abundance, GLUT1 trafficking, and GLUT1's activity toward its two substrates in skeletal muscle. Preliminary data suggest that ataxia telangiectasia mutated (ATM) may play a role in regulation of basal glucose transport and GLUT1 abundance in skeletal muscle. In addition, other investigators have reported a potential role for p38 in increasing intrinsic activity of GLUT1 toward glucose transport. There are three C-terminal phosphorylation sites in GLUT1;S490 is known target of ATM, and S473 and T478 match consensus target motifs for ATM and p38, respectively. The goal of the project is to determine the roles of these GLUT1 phosphorylation sites (or activation of the potential kinases for these sites) in regulation of GLUT1 abundance, localization, and activity in cultured skeletal muscle cells or mouse skeletal muscle. Specific Aim 1 of the project is to determine whether S473 and S490 of GLUT1 play roles in regulation of GLUT1 abundance or cell surface localization. The general hypotheses are that phosphorylation of these sites will preserve GLUT1 levels and cell surface localization, while GLUT1 that is not phosphorylated at these sites will be more prone toward internalization and degradation. Effects of S473 and S490 mutations will be determined for GLUT1 abundance, trafficking, and transport activity toward glucose and DHA, and effects of activation of ATM (a potential kinase for both sites) on GLUT1 will also be determined. Specific Aim 2 is to determine whether T478 plays a role in regulation of substrate specificity or intrinsic activity of GLUT1. The hypothesis is that T478 phosphorylation will stimulate increased GLUT1 activity toward glucose and decrease activity toward DHA. Effects of T478 mutations will be determined for intrinsic activity of GLUT1 toward glucose and DHA, though potential effects on GLUT1 abundance and trafficking will also be examined. Effects of activation of p38 (a potential kinase of T478) on GLUT1 activity and trafficking will be determined. Additionally, it will be determined whether factors that alter DHA transport also are associated with changes in levels of reactive oxygen species and/or can influence insulin action in muscle cells. Information provided by this project might be used to develop strategies to increase basal glucose transport in skeletal muscle or to increase DHA transport to support skeletal muscle antioxidant status. PUBLIC HEALTH RELEVANCE: This project will investigate factors that could regulate GLUT1, a protein that allows movement of sugar into skeletal muscle all day long (as opposed to another sugar-transporting protein that is responsible for moving sugar into muscle after meals or exercise). Additionally, the role of GLUT1 in providing the building block for muscle vitamin C, a key antioxidant that destroys free radicals, will be investigated. The goal of the project is to provide information that might be useful in improving blood sugar control or in maintaining antioxidant defenses in muscle.